Bishispidine compounds, processes for their production and pharmaceutical agents containing these compounds

ABSTRACT

The invention concerns new bishipidine derivatives, processes for their production and pharmaceutical agents containing these compounds. The new methylene-bishispidine derivatives can be used for the prophylaxis and therapy of late diabetic damage as well as for the prophylaxis and therapy of atherosclerosis and arteriosclerosis.

This application is a 371 of PCT/EP94/03116 filed Sep. 17, 1994,published as WO95/08547, Mar. 30, 1995.

The present invention concerns methylene-bishispidine derivatives,processes for their production and pharmaceutical agents that containthese compounds.

The invention concerns methylene-bishispidine derivatives of the generalformula I ##STR1## in which A denotes hydrogen, C₁ -C₁₆ alkyl, C₃ -C₆cycloalkyl, a group ##STR2## in which R₁ -R₅ simultaneously orindependently of one another denote hydrogen, C₁ -C₆ alkyl, hydroxy, C₁-C₆ acyloxy, C₁ -C₆ alkoxy, benzyloxy, halogen, cyano, carboxy, amino,phenyl, alkylthio, carboxyalkyl, heteroalkyl or an ester group,

X is --CR₆ ═CR₇ --, --CR₈ ═N--, --N═CR₉ --, oxygen or sulphur,

Y is --CR₆ ═CR₇ --, --CR₁₁ ═N--, NR₁₀, oxygen or sulphur

Z is ═CR₁₁ -- or nitrogen

in which

R₆ -R₁₁ independently of one another denote hydrogen, methyl, halogen orcarboxy and

W denotes hydrogen, C₁ -C₄ alkyl, optionally substituted by halogen,arylalkyl, carboxyl or carboxyl ester

as well as physiologically tolerated salts or esters thereof.

The alkyl residues in the said alkyl, alkoxy, acyloxy, alkylthio,carboxyalkyl or heteroalkyl groups can be straight-chained or branched.A C₁ -C₆ alkyl residue in the present invention denotes methyl, ethyl,propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl or hexyl.Heteroalkyl denotes a C₁ -C₄ alkyl residue substituted by nitrogen,oxygen or sulphur. Arylalkyl denotes a phenyl group which is optionallysubstituted by halogen, hydroxy or methyl and is linked by a C₁ -C₄alkyl residue. Carboxyalkyl is a carboxy group linked by a C₁ -C₄ alkylresidue. Halogen denotes fluorine, chlorine, bromine or iodine,preferably chlorine.

It was surprisingly found that compounds of formula I have valuablepharmacological properties. In particular they inhibit the formation ofAGE (Advanced Glycosylation Endproducts) whose significance for thedevelopment of late diabetic complications has been shown (A. Cerami,Trends Biochem. Sci. 11, 311 (1986)).

Thus the non-enzymatic glycosylation of plasma proteins can bestimulated in vitro by incubating them with glucose the reinjection ofthese proteins leads in vivo to typical late diabetic damage (H.Vlassara et al., Diabetes 41, Suppl. 1, 9A (1992)). AGE are involved inthe thickening of the glomerular basal membrane, a process which isresponsible for renal insufficiency and renal failure. The non-enzymaticglycosylation of crystallin, a protein of the eye lens, leads to changesin the tertiary structure and polymerization by oxidation of SH groupsto disulfides resulting in diabetic cataract formation (V. Monnier,Clin. Endocrinol. Metab. 11, 431 (1982)). The cross-linking of proteinscaused by the end products of non-enzymatic glycosylation reduces thesolubility of collagen and is involved in the sclerosis of blood vessels(H. Rosenburg et al., Biochem. Biophys. Res. Commun. 91 498 (1979)). Afurther consequence is the capture of low density lipoproteins (M.Brownlee et al., Science 232, 1629 (1986)). The localization of theseLDL proteins on the endothelium is a strong stimulus for atheroscleroticprocesses (D. Steinberg et al., J. Clin. Invest, 88, 1785 (1991); D.Leake, Current Opinion in Lipidology, 2, 301 (1991)).

Compounds of formula I are therefore suitable for the prophylaxis andtreatment of late diabetic damage (e.g. retinopathy, nephropathy andneuropathy) as well as for the prophylaxis and therapy ofatherosclerosis and arteriosclerosis.

Preferred compounds of the general formula I are compounds in which Adenotes hydrogen, C₁ -C₆ -alkyl, C₃ -C₆ cycloalkyl or a group ##STR3##in which R₁ and R₅ are hydrogen.

Particularly preferred compounds of the general formula I are those inwhich R³ represents hydrogen, hydroxy, C₁ -C₄ alkoxy, cyano, carboxy orhalogen, R² and R⁴ independently of one another and independently of R³denote hydrogen, hydroxy, C₁ -C₄ alkyloxy or C₁ -C₄ alkyl and R¹ and R⁵are hydrogen atoms.

Particularly preferred compounds of the general formula I are also thosein which A denotes a group ##STR4## in which X is --CH═CH--, NH, --CH═N,oxygen or sulphur,

Y is --CH═CH--, --CH═N-- or sulphur and

Z is ═CH-- or nitrogen.

W in the general formula I is preferably hydrogen, C₁ -C₄ alkyl or acarboxyl ester.

The compounds of the general formula I in which A and W have themeanings stated above are produced by condensing hispidine (II) with analdehyde or a ketone of formula III ##STR5## in this case A and W havethe above-mentioned meanings. The reaction can be carried out by heatingthe two components in a polar organic solvent, preferably in proticsolvents such as simple aliphatic alcohols, in particular methanol orethanol, in the presence of catalytic amounts of a mineral acid such ashydrochloric acid or sulphuric acid. The reaction can also be carriedout in a dipolar aprotic solvent such as dimethyl-formamide withaddition of piperidinium acetate as a catalyst with azeotropicdehydration with toluene.

If the synthesized compounds of the general formula I are acidic orbasic they can, if desired, be converted into physiologically toleratedsalts and in the case of carboxylic acids it is possible to convert theminto esters using physiologically acceptable alcohols. Pharmacologicallyacceptable inorganic or organic bases such as e.g. sodium hydroxide,potassium hydroxide, calcium hydroxide, methylene glucamine, morpholineor ethanolamine are suitable for the formation of salts from carboxylicacids of the general formula I. Acids which are suitable for formingsalts with bases of the general formula I are for example hydrochloricacid, sulphuric acid, acetic acid, citric acid, maleic acid, fumaricacid and tartaric acid.

If the compounds of the general formula I contain a carboxyl function,esters of these carboxylic acids which come into consideration are thoseformed with lower monovalent alcohols (e.g. methanol or ethanol) or withpolyvalent alcohols (such as e.g. glycerol).

The starting compound hispidine (II) used for the production of thecompounds of formula I was discovered and structurally characterizedalmost simultaneously by R. L. Edwards et al. (isolation from Polyporushispidus; J. chem. Soc. 1961, 4995) and A. Ueno et al. (isolation fromPhaeolus schweinitzii"; Chem. Pharm. Bull. 12, 376 (1964)).

However, the amounts of hispidine which can be obtained by extractionfrom basidiomycetes are much too small to serve as the starting materialfor extensive syntheses and in-depth pharmacological investigations.Consequently there was an urgent need to develop an efficient chemicalproduction process for hispidine.

A first but not very efficient hispidine synthesis was described in 1961by Edwards et al. In this process 3,4-di(methoxymethoxy)benzaldehyde isreacted with 4-methoxymethoxy-6-methyl-2-pyrone in the presence ofmagnesium methanolate in methanol, the yield being only 2% hispidinesince an extremely complex mixture of products is formed.

The reaction of 3,4-di(methoxymethoxy)benzaldehyde with4-methoxy-6-methyl-2-pyrone in the presence of magnesium methanolate inmethanol is stated as an alternative in the publication cited above. Thesubsequent alkaline saponification and acidic recyclization leads tosuch a complex reaction mixture that hispidine that is formed can onlybe isolated after acetylation as a tri-O-acetyl compound (5% yield). Theisolation of the free hispidine then requires yet a further stepinvolving heavy losses.

In both the processes described by Edwards et al the preparation of thestarting materials used in addition requires the use of the highlycarcinogenic chloromethyl ether which practically excludes an industrialapplication.

The subject matter of the present invention is thus a process which isbased on the reaction of 3,4-dibenzyloxy-benzaldehyde IV with4-benzyloxy-6-methyl-2-pyrone V in the presence of lithiumdiisopropylamide. The aldol compound VI formed can be debenzylated undervery mild conditions by catalytic hydrogenation to form the tetrahydroxycompound VII. It is readily dehydrated to hispidine II with dilute acid.The process according to the invention has a very reproducible totalyield (IV→II) of 30% and is thus considerably superior to the previouslyknown methods for the preparation of hispidine.

For the production of pharmaceutical agents the compounds of the generalformula I are mixed with suitable pharmaceutical carrier substances,aromatics, flavourings and dyes in a well-known manner and are forexample formed into tablets or dragees or suspended or dissolved inwater or oil such as e.g. olive oil with addition of correspondingauxiliary agents.

The methylene-bishispidine derivatives of the general formula I can beadministered orally and parenterally in a liquid or solid form. Water ispreferably used as the injection medium which contains the usualstabilizing agents, solubilizers and/or buffers for injection solutions.Such additives are for example tartrate or borate buffer, ethanol,dimethyl-sulfoxide, complexing agents (such asethylenediamine-tetraacetic acid), high molecular polymers (such asliquid polyethylene oxide) to regulate the viscosity or polyethylenederivatives of sorbitol anhydrides.

Solid carriers are e.g. starch, lactose, mannitol, methylcellulose,talcum, highly dispersed silicic acid, higher molecular polymers (suchas polyethylene glycols). Preparations that are suitable for oraladministration can if desired contain flavourings and sweeteners.

The administered dose depends on the age, state of health and weight ofthe recipient, the extent of the disease, the type of further treatmentsthat are simultaneously carried out if desired and the type of thedesired effect. The daily dose of the active compound is usually 0.1 to50 mg/kg body weight. 0.5 to 40 and preferably 1.0 to 20 mg/kg/day inone or several administrations per day are usually effective in order toobtain the desired results.

Within the sense of the present invention the following compounds offormula I come into consideration in addition to the compounds mentionedin the examples and compounds derived by combination of all meanings ofsubstituents mentioned in the claims, which can be present optionally assalts or esters.

1. 3,3'-(4-Hydroxybenzylidene)bis( 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!

2. 3,3'-(2-Pyrrolylmethylene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!

3. 3,3'-(2-Phenylethylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2 -one!

4. 3,3'-(4-Carboxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!

Examples of application

Synthesis of hispidine (II):

i) 4-Benzyloxy-6-methyl-2H-pyran-2-one (V)

124.4 g (0.900 mol) potassium carbonate and 63 ml (0.547 mol) benzylchloride were added in succession to a solution of 56.7 g (0.450 mol)4-hydroxy-6-methyl-2H-pyran-2-one and 1.0 g (0.004 mol) 18-crown-6 in900 ml anhydrous dimethyl-formamide. It was stirred vigorously for 4.5hours at an internal temperature of 70°-75° C. It was suction filteredand the residue was washed well with ethyl acetate. The combinedfiltrates were extensively evaporated in a vacuum and subsequentlypoured onto ice water. The aspirated precipitate was recrystallized fromt-butylmethyl ether after drying. 69.0 g (71%) V, melting point 92°-93°C.

ii) 4-Benzyloxy-6-2-(3,4-dibenzyloxyphenyl)-2-hydroxyethyl!-2H-pyran-2-one (VI)

A solution of 10.1 g (14.0 ml, 100 mmol) diisopropylamine in 150 mltetrahydrofuran was mixed under nitrogen at -78° C. with 41.0 ml (95mmol) butyllithium (2.3M in n-hexane). It was allowed to reach 0° C. fora short time and immediately cooled again to -78° C. After a dropwiseaddition of a solution of 19.0 g (88 mmol)4-benzyloxy-6-methyl-2H-pyran-2-one (V) in 100 ml anhydroustetrahydrofuran it was stirred for a further 45 min. at -78° C. andsubsequently a solution of 12.8 g (40 mmol) 3,4-dibenzyloxy-benzaldehyde(IV) in 150 ml anhydrous tetrahydro-furan was added dropwise. Theinternal temperature was gradually increased to -30° C. and it wasadmixed with an excess of saturated ammonium chloride solution. Afteracidifying with 6n HCl it was extracted with ethyl acetate, the combinedorganic phases were dried and evaporated. Purification of the residue bymeans of flash chromatography on silica gel (mobile solvent: ethylacetate/heptane 1:2) yielded 14.4 g (68%) VI, melting point 109°-110° C.(ether).

iii) 4-Hydroxy-6- 2-(3,4-dihydroxyphenyl)-2-hydroxyethyl!-2H-pyran-2-one(VII)

35.6 g (66.6 mmol) 4-benzyloxy-6- 2-3,4-dibenzyloxyphenyl)-2-hydroxy-ethyl)!-2H-pyran-2-one (VI) washydrogenated for 6 hours at room temperature under a hydrogen pressureof 44 mbar in 1200 ml ethanol using 10 g W2 Raney nickel. After takingup 4.4 l hydrogen the catalyst was removed by suction filtration and itwas rewashed several times with tetrahydrofuran. The evaporatedfiltrates yielded 12.2 g (70%) VII after triturating with ether, meltingpoint 153° C. (decomp.).

iv) 6- 2-(3,4-Dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one(hispidine), (II)

2 ml 3N HCl was added to a suspension of 8.0 g (30 mmol) 4-hydroxy-6-2-(3,4-dihydroxyphenyl)-2-hydroxyethyl!-2H-pyran-2-one VII and 300 mltetrahydrofuran. The resulting clear solution was heated for 2-3 hoursunder reflux until it was no longer possible to detect startingmaterial. (TLC control, mobile solvent: toluene/ethyl formate/methanol5:4:1, 0.5% formic acid). After drying over sodium sulphate the solutionwas strongly evaporated (residual volume ca. 50 ml) and it wascrystallized by admixing with ether. Yield 4.7 g (63%) hispidine II,melting point 232°-234° C. (decomp.) or melting point 253° C. (decomp;bunch of needles from toluene/ethyl formate).

Synthesis of methylene-bishispidine derivatives of the general formulaI:

EXAMPLE 1

3,3'-(4-Chlorobenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!

4.9 g (0.02 mol) hispidine was added to a solution of 7.0 g (0.05 mol)4-chlorobenzaldehyde in 100 ml methanol and it was subsequently admixedwith 2.0 ml hydrochloric acid. The mixture was stirred at 50° C. untilthe reaction was completed (progress monitored by TLC, mobile solvent:toluene/ethyl formate/formic acid 5:4:1). The reaction mixture wasstirred into 500 ml water and the brown precipitate was aspirated (crudeyield 6.1 g). For the purification it was precipitated from ethylacetate/heptane. 3.1 g (50%), melting point 193° C. (decomp.).

EXAMPLE 2

3,3'-(4-Methylenebenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!

Analogously to example 1 the compound obtained from hispidine and4-methylbenzaldehyde was stirred with ether after purification of theaspirated crude product by flash chromatography on silica gel (mobilesolvent: toluene/dioxane/glacial acetic acid 72:20:8). Yield 43%,melting point 192° C. (decomp.).

EXAMPLE 3

3,3'-(3-Hydroxy-4-methoxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one! (3)

Analogously to example 2 from hispidine and3-hydroxy-4-methoxybenzaldehyde. Mobile solvent: toluene/ethylformate/methanol 5:4:1, 0.5% formic acid. Yield: 36%, melting point 181°C. (decomp.).

EXAMPLE 4

3,3'-(4-Hydroxy-3,5-(bis-1,1-dimethylethyl) benzylidene!bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one! (4)

Analogously to example 2 from hispidine and4-hydroxy-3,5-(bis-1,1-di-methylethyl)-benzaldehyde. Mobile solvent:toluene/dioxane/glacial acetic acid 72:20:8) Yield: 40%, melting point148° C. (decomp.).

EXAMPLE 5

3,3'-(4-Methoxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (5)

Analogously to example 2 from hispidine and 4-methoxybenzaldehyde.Mobile solvent toluene/ethyl formate/methanol 5:4:1, 1% formic acid.Yield 28%, melting point 167° C. (decomp.).

EXAMPLE 6

3,3'-(3,4-Dimethoxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one!

Analogously to example 2 from hispidine and 3,4-dimethoxy-benzaldehyde.Mobile solvent toluene/ethyl formate/methanol 5:4:1, 0.5% formic acid.Yield 32%, melting point 177° C. (decomp.).

EXAMPLE 7

3,3'-(3,4-Dihydroxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (7)

Analogously to example 2 from hispidine and 3,4-dihydroxy-benzaldehyde.Mobile solvent toluene/ethyl formate/methanol 5:4:1, 1% formic acid.Yield 41%, melting point 238° C. (decomp.).

EXAMPLE 8

3,3'- 4-Cyanobenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (8)

Analogously to example 1 from hispidine and 4-cyanobenz-aldehyde.Recrystallized from ethyl acetate/heptane. Yield 41%, melting point 198°C. (decomp.).

EXAMPLE 9

3,3'-Benzylidenebis 6-2-(3,4-dihydroxypheny1)vinyl-4-hydroxy-2H-pyran-2-one! (9)

Analogously to example 2 from hispidine and benzaldehyde. Mobile solventtoluene/ethyl formate/methanol 5:4:1, 0.5% formic acid. Yield 33%,melting point 163° C. (decomp.).

EXAMPLE 10

3,3'-Methylenebis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (10)

Analogously to example 2 from 1.00 g (4.00 mmol) hispidine and 15 ml(0.2 mol) 35% formalin solution. Re-precipitated fromtetrahydrofuran/isohexane. Yield 28%, melting point 290° C. (decomp.).

EXAMPLE 11

3,3'-(2-Thienylmethylene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (11)

Analogously to example 2 from hispidine and thiophene-2-aldehyde. Mobilesolvent toluene/ethyl formate/methanol 5:4:1, 0.5% formic acid. Yield31%, melting point>300° C.

EXAMPLE 12

3,3'-(2-Pyridylmethylene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one!hydrochloride (12)

Analogously to example 1 from hispidine and pyridine-2-aldehyde. Theaspirated precipitate was intensively washed with ethyl acetate. Yield55%, melting point 228° C. (decomp.).

EXAMPLE 13

3,3'-(3,4,5-Trimethoxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one!

Analogously to example 2 from hispidine and3,4,5-trimethoxybenzaldehyde. Mobile solvent: toluene/ethylformate/methanol 5:4:1, 0.5% formic acid. Yield 30%, melting point 208°C. (decomp.).

EXAMPLE 14

3,3'-(3-Thienylmethylene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (14)

Analogously to example 2 from hispidine and thiophene-3-aldehyde. Mobilesolvent: toluene/ethyl formate/methanol 5:4:1, 0.5% formic acid. Yield35%, melting point 243° C. (decomp.).

EXAMPLE 15

3,3'-(1-Hexylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one!

Analogously to example 2 from 1.00 g (4.00 mmol) hispidine and 9.6 ml(80 mmol) hexanal. Mobile solvent: toluene/ethyl formate/methanol 5:4:1,0.5% formic acid. Yield 45%, melting point 109° C. (decomp.).

EXAMPLE 16

3,3'-(2-Imidazolylmethylene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one!hydrochloride (16)

Analogously to example 2 from hispidine and imidazole-2-aldehyde. Mobilesolvent: toluene/ethyl formate/methanol 5:4:1, 0.5% formic acid. Yield4%, melting point 147° C. (decomp.).

EXAMPLE 17

3,3'-(4-Bromobenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (17)

Analogously to example 2 from hispidine and 4-bromobenz-aldehyde. Mobilesolvent: toluene/ethyl formate/methanol 5:4:1, 0.5% formic acid. Yield27%, melting point 228° C. (decomp.).

EXAMPLE 18

3,3'-(4-Benzyloxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (18)

Analogously to example 2 from hispidine and 4-benzyloxy-benzaldehyde.Mobile solvent: toluene/ethyl formate/methanol 5:4:1, 0.5% formic acid.Yield 11%, melting point 180° C. (decomp.).

EXAMPLE 19

3,3'-Methoxycarbonylmethylenebis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (19)

Analogously to example 2 from hispidine and glyoxylic acid. Mobilesolvent: toluene/ethyl formate/methanol 5:4:1, 0.5% formic acid. Yield18%, melting point 203° C. (decomp.).

EXAMPLE 20

3,3'-(Cyclohexylmethylene)bis 6-2-(3,4-dihydroxyphenyl)vinyl-4-hydroxy-2H-pyran-2-one! (20)

Analogously to example 2 from 1.00 g (4.00 mmol) hispidine and 6.7 g (60mmol) cyclohexanaldehyde, purification of the aspirated precipitate byflash chromatography on silica gel (mobile solvent: toluene/ethylformate/methanol 5:4:1, 0.5% formic acid. Yield 36%, melting point 154°C. (decomp.).

DESCRIPTION OF THE EXPERIMENT

Test for the inhibitory activity on the formation of AGE

Lysozyme is dissolved at a final concentration of 20 mg/ml in 100 mmol/lpotassium phosphate buffer, pH 7.4. 5 mmol/l sodium azide is added as apreservative. The formation of AGE is initiated by the addition of 200mmol/l glucose or xylose. Test substances or the reference substanceaminoguanidine are added at different concentrations in order toquantify an inhibitory activity on AGE formation. Solvent controls aswell as control mixes are also run in the presence of 200 mmol/lsorbitol or xylite for correction purposes. The incubation period of thereaction mixtures is between 24 and 120 hours depending on the sugarused and is selected in such a way that mainly dimers are formed andother higher molecular oligomerization products are formed only to aslight extent.

The samples are prepared and SDS gel electrophoresis is carried outaccording to literature references. Summarized briefly the samples arediluted with the 4-fold amount of sample buffer (50 mmol/l Tris^(*)acetate, pH 7.5, 1% SDS, 5 mmol/l dithiothreitol) and heated for 3minutes to 95° C. Aliquots are applied to 8-18% gradient-SDS gels. Inorder to determine the molecular weight molecular weight markers arealso used in the range 14-340 kD. The separation according to molecularweight is achieved under the following operating conditions: voltage:600 V, current intensity: 50 mA, power: 30 W, run period: ˜60 minutes.

After the gel electrophoresis the gels are fixed according to standardmethods and stained with Coomassie Blue. After destaining overnight anddrying, the gels are measured densitometrically (Pharmacia Ultra Scan XLusing GelScan XL software).

In order to determine the threshold concentration and the IC₅₀ values ofthe AGE-inhibitory action of the test substances, the dimer formation isplotted semi-logarithmically against the test concentration of thesubstance and the above-mentioned parameters are calculated.

                  TABLE                                                           ______________________________________                                        Compound      Threshold concentration                                                                      IC.sub.50                                        Example No.    μmol/l!     μmol/l!                                      ______________________________________                                        1             >1             4                                                3             >1             2                                                4             >1             2                                                5             >1             2                                                6             >1             5                                                7             2              4                                                8             10             22                                               aminoguanidine                                                                              500            4500                                             ______________________________________                                    

We claim:
 1. A methylene-bishispidine compound of the formula ##STR6##wherein A is hydrogen, C₁ -C₁₆ alkyl, C₃ -C₆ cycloalkyl or a group ofthe formula ##STR7## wherein R₁ -R₅ are independently hydrogen, C₁ -C₆alkyl, hydroxy, C₁ -C₆ acyloxy, C₁ -C₆ alkoxy, benzyloxy, halogen,cyano, carboxy, amino, phenyl, alkylthio, carboxy-C₁ -C₄ -alkyl, C₁ -C₄alkyl substituted by nitrogen, oxygen or sulphur, or an ester of a C₁-C₆ carboxylic acid with a C₁ -C₄ alcohol or a C₁ -C₄ polyol;X is --CR₆═CR₇ --, --CR₈ ═N--, --N═CR₉ --, oxygen or sulphur, Y is --CR₆ ═CR₇ --,--CR₁₁ ═N--, --NR₁₀ --, oxygen or sulphur, Z is ═CR₁₁ -- or nitrogen,wherein R₆ -R₁₁ are independently hydrogen, methyl, halogen or carboxy;and W is hydrogen, C₁ -C₄ alkyl which is unsubstituted or substituted byhalogen, phenyl-C₁ -C₄ -alkyl wherein the phenyl ring is unsubstitutedor substituted by halogen, hydroxy or methyl, carboxyl or an ester of aC₁ -C₆ carboxylic acid with a C₁ -C₄ alcohol or a C₁ -C₄ polyol; or aphysiologically tolerated salt or ester thereof.
 2. Amethylene-bishispidine compound of claim 1 whereinA is hydrogen, C₁ -C₆alkyl, C₃ -C₅ cycloalkyl, or a group of the formula ##STR8## wherein R₁and R₅ are hydrogen.
 3. A methylene-bishispidine compound of claim 2,whereinR₃ is hydrogen, hydroxy, C₁ -C₄ alkoxy, cyano, carboxy orhalogen, and R₂ and R₄ are independently hydrogen, hydroxy, C₁ -C₄alkoxy or C₁ -C₄ alkyl.
 4. A methylene-bishispidine compound as claimedin claim 1, wherein A is a group of the formula ##STR9## wherein X is--CH═CH--, --NH--, --CH═N--, oxygen or sulphur;Y is --CH═CH--, --CH═N--or sulphur; and Z is ═CH-- or nitrogen.
 5. A methylene-bishispidinecompound of claim 1, wherein W is hydrogen, C₁ -C₄ alkyl or a carboxylester.
 6. A methylene-bishispidine compound of claim 1, wherein saidderivative is:3-3'-(4-Hydroxybenzylidene)bis(6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!,3,3'-(2-Pyrrolylmethylene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!,3,3'-(2-Phenylethylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-one!,3,3'-(4-Carboxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy -2 H-pyran-one!,3,3'-(4-Chlorobenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!,3,3'-(Methoxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!,3,3'-(3-Hydroxy-4-methoxybenzylidene)bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!, 3.3'-(4-Hydroxy-3,5-(bis-1,1-dimethylethyl) benzylidene!bis 6-2-(3,4-dihydroxyphenyl)vinyl!-4-hydroxy-2H-pyran-2-one!3,3'-(4-Methoxybenzylidene)bis6- 2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(3,4-Dimethoxybenzylidene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(3,4-Dimethoxybenzylidene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(4-Cyanobenzylidene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-Benzylidenebis 6- 2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!, 3,3'-Methylenebis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(2-Thienylmethylene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(2-Pyridylmethylene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!hydrochloride,3,3'-(3,4,5,-Trimethoxybenzylidene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(3-Thienylmethylene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(1-Hexylidene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(2-Imidazolylmethylene)bis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!hydrochloride, 3.3'-(4-Bromobenzylidenebis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!,3,3'-(4-Benzyloxybenzylidene)bis6- 2-(3,4-dihydroxphenyl)vinyl-4-hydroxy -2 H-pyran-2-one!,3,3'-Methoxycarbonylmethylenebis 6-2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2H-pyran-2-one!, or3,3'-(Cyclohexylmethylene)bis 6- 2-(3,4-dihydroxphenyl)vinyl-4-hydroxy-2 H-pyran-2-one!.
 7. A pharmaceutical composition suitable for theinhibition of the formation of Advanced Glycosylation Endproductscomprising an effective amount of a methylene-bishispidine compound ofclaim 1 and a pharmaceutically acceptable carrier therefor.
 8. A methodfor the treatment of late diabetic damage, atherosclerosis orarteriosclerosis in a patient in need of such treatment, comprisingadministering to the patient an effective amount of amethylene-bishispidine compound of claim
 1. 9. A method of inhibitingthe formation of Advanced Glycosylation Endproducts (AGE) in a patientin need of such inhibition, comprising administering to the patient anAGE-inhibiting effective amount of a methylene-bishispidine compound ofclaim
 1. 10. A process for producing a methylene-bishispidine derivativeof claim 1, comprising reacting hispidine of the formula ##STR10## withan aldehyde or ketone of the formula ##STR11## wherein A and W aredefined in claim 1, with heating in a polar organic solvent in thepresence of a catalytic amount of a mineral acid or in a dipolar aproticsolvent in the presence of a catalytic amount of piperidinium acetateunder azeotropic dehydration.
 11. A process according to claim 10,wherein the hispidine is produced by reacting3,4-dibenzyloxybenzaldehyde of the formula: ##STR12## with4-benzyloxy-6-methyl-2H-pyran-2-one of the formula: ##STR13## in thepresence of lithium diisopropylamide to form an aldol compound of theformula: ##STR14## thereafter debenzylating the aldol compound to form atetrahydroxy compound of the formula: ##STR15## and thereafterdehydrating the tetrahydroxy compound to form hispidine.